Human cathepsin G lacking functional glycosylation site is proteolytically processed and targeted for storage in granules after transfection to the rat basophilic/mast cell line RBL or the murine myeloid cell line 32D
(1995) In The Journal of biological chemistry 270(47). p.8-28413- Abstract
The neutral protease cathepsin G belongs to a family of hematopoietic serine proteases stored in the azurophil granules of the neutrophil granulocyte. To investigate the function of asparagine-linked carbohydrates in neutrophil serine proteases, we constructed a mutant cDNA, coding for human cathepsin G deficient of a functional glycosylation site, for use in a transgenic cellular model. Wild type and mutant cDNA were stably expressed in the rat basophilic/mast cell line RBL and in the murine myeloblast-like cell line 32D. Biosynthetic labeling, followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography, showed that carbohydrate-deficient cathepsin G was synthesized as a 29-kDa proform in both cell lines.... (More)
The neutral protease cathepsin G belongs to a family of hematopoietic serine proteases stored in the azurophil granules of the neutrophil granulocyte. To investigate the function of asparagine-linked carbohydrates in neutrophil serine proteases, we constructed a mutant cDNA, coding for human cathepsin G deficient of a functional glycosylation site, for use in a transgenic cellular model. Wild type and mutant cDNA were stably expressed in the rat basophilic/mast cell line RBL and in the murine myeloblast-like cell line 32D. Biosynthetic labeling, followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography, showed that carbohydrate-deficient cathepsin G was synthesized as a 29-kDa proform in both cell lines. The proform was proteolytically processed into a stable form with an apparent molecular mass of 27.5 kDa, indicating removal of the carboxyl-terminal prodomain. The mutant cathepsin G was enzymatically activated as determined by acquisition of affinity to aprotinin, a serine protease inhibitor. As for wild type cathepsin G, small amounts of the unprocessed form of the mutated enzyme were released from the cells, while the major part was transferred to a granular compartment as demonstrated by subcellular fractionation. Thus, neither processing leading to enzymatic activation nor granular sorting was obviously affected by the lack of oligosaccharides on the mutant cathepsin G. Our results therefore indicate that glycosylation is not essential for these processes. In addition to the previously utilized cell line RBL, we propose the 32D cell line as a suitable cellular model for transgenic expression of human neutrophil serine proteases.
(Less)
- author
- Garwicz, Daniel LU ; Lindmark, Anders and Gullberg, Urban LU
- organization
- publishing date
- 1995-11-24
- type
- Contribution to journal
- publication status
- published
- keywords
- Amino Acid Sequence, Animals, Base Sequence, Cathepsin G, Cathepsins/biosynthesis, Cell Line, Chromatography, Affinity, Cytoplasmic Granules/metabolism, DNA Primers, Glutamine, Glycosylation, Humans, Kinetics, Leukemia, Basophilic, Acute, Mast Cells, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Point Mutation, Polymerase Chain Reaction, Protein Processing, Post-Translational, Rats, Recombinant Proteins/biosynthesis, Sequence Deletion, Serine Endopeptidases, Transfection, Tumor Cells, Cultured
- in
- The Journal of biological chemistry
- volume
- 270
- issue
- 47
- pages
- 8 - 28413
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- scopus:0028845274
- pmid:7499346
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.270.47.28413
- language
- English
- LU publication?
- yes
- id
- 77a8b78b-d5f4-422f-b25a-c8eb2a6536fc
- date added to LUP
- 2025-02-28 12:35:46
- date last changed
- 2025-03-01 04:01:33
@article{77a8b78b-d5f4-422f-b25a-c8eb2a6536fc,
abstract = {{<p>The neutral protease cathepsin G belongs to a family of hematopoietic serine proteases stored in the azurophil granules of the neutrophil granulocyte. To investigate the function of asparagine-linked carbohydrates in neutrophil serine proteases, we constructed a mutant cDNA, coding for human cathepsin G deficient of a functional glycosylation site, for use in a transgenic cellular model. Wild type and mutant cDNA were stably expressed in the rat basophilic/mast cell line RBL and in the murine myeloblast-like cell line 32D. Biosynthetic labeling, followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography, showed that carbohydrate-deficient cathepsin G was synthesized as a 29-kDa proform in both cell lines. The proform was proteolytically processed into a stable form with an apparent molecular mass of 27.5 kDa, indicating removal of the carboxyl-terminal prodomain. The mutant cathepsin G was enzymatically activated as determined by acquisition of affinity to aprotinin, a serine protease inhibitor. As for wild type cathepsin G, small amounts of the unprocessed form of the mutated enzyme were released from the cells, while the major part was transferred to a granular compartment as demonstrated by subcellular fractionation. Thus, neither processing leading to enzymatic activation nor granular sorting was obviously affected by the lack of oligosaccharides on the mutant cathepsin G. Our results therefore indicate that glycosylation is not essential for these processes. In addition to the previously utilized cell line RBL, we propose the 32D cell line as a suitable cellular model for transgenic expression of human neutrophil serine proteases.</p>}},
author = {{Garwicz, Daniel and Lindmark, Anders and Gullberg, Urban}},
issn = {{0021-9258}},
keywords = {{Amino Acid Sequence; Animals; Base Sequence; Cathepsin G; Cathepsins/biosynthesis; Cell Line; Chromatography, Affinity; Cytoplasmic Granules/metabolism; DNA Primers; Glutamine; Glycosylation; Humans; Kinetics; Leukemia, Basophilic, Acute; Mast Cells; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction; Protein Processing, Post-Translational; Rats; Recombinant Proteins/biosynthesis; Sequence Deletion; Serine Endopeptidases; Transfection; Tumor Cells, Cultured}},
language = {{eng}},
month = {{11}},
number = {{47}},
pages = {{8--28413}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{The Journal of biological chemistry}},
title = {{Human cathepsin G lacking functional glycosylation site is proteolytically processed and targeted for storage in granules after transfection to the rat basophilic/mast cell line RBL or the murine myeloid cell line 32D}},
url = {{http://dx.doi.org/10.1074/jbc.270.47.28413}},
doi = {{10.1074/jbc.270.47.28413}},
volume = {{270}},
year = {{1995}},
}